US20060182735A1 - Thrombin preparations and process for their production - Google Patents
Thrombin preparations and process for their production Download PDFInfo
- Publication number
- US20060182735A1 US20060182735A1 US11/385,713 US38571306A US2006182735A1 US 20060182735 A1 US20060182735 A1 US 20060182735A1 US 38571306 A US38571306 A US 38571306A US 2006182735 A1 US2006182735 A1 US 2006182735A1
- Authority
- US
- United States
- Prior art keywords
- thrombin
- mmol
- preparation
- hydrophobic interaction
- interaction chromatography
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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- 108090000190 Thrombin Proteins 0.000 title claims abstract description 115
- 229960004072 thrombin Drugs 0.000 title claims abstract description 110
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 5
- 238000000034 method Methods 0.000 title claims description 37
- 230000008569 process Effects 0.000 title claims description 35
- 238000004191 hydrophobic interaction chromatography Methods 0.000 claims abstract description 19
- 241000700605 Viruses Species 0.000 claims abstract description 15
- 108010094028 Prothrombin Proteins 0.000 claims abstract description 13
- 102100027378 Prothrombin Human genes 0.000 claims abstract description 13
- 229940039716 prothrombin Drugs 0.000 claims abstract description 13
- 238000005277 cation exchange chromatography Methods 0.000 claims abstract description 12
- 239000003381 stabilizer Substances 0.000 claims abstract description 11
- 239000003112 inhibitor Substances 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 40
- 230000000694 effects Effects 0.000 claims description 24
- 239000011780 sodium chloride Substances 0.000 claims description 20
- 238000001914 filtration Methods 0.000 claims description 8
- 239000002253 acid Substances 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 230000002209 hydrophobic effect Effects 0.000 claims description 5
- 230000002779 inactivation Effects 0.000 claims description 5
- 230000009467 reduction Effects 0.000 claims description 5
- WPANETAWYGDRLL-UHFFFAOYSA-N 4-aminobenzenecarboximidamide Chemical compound NC(=N)C1=CC=C(N)C=C1 WPANETAWYGDRLL-UHFFFAOYSA-N 0.000 claims description 4
- 150000001413 amino acids Chemical class 0.000 claims description 4
- PXXJHWLDUBFPOL-UHFFFAOYSA-N benzamidine Chemical group NC(=N)C1=CC=CC=C1 PXXJHWLDUBFPOL-UHFFFAOYSA-N 0.000 claims description 4
- 159000000007 calcium salts Chemical class 0.000 claims description 4
- 230000002439 hemostatic effect Effects 0.000 claims description 4
- 239000012528 membrane Substances 0.000 claims description 4
- 239000003106 tissue adhesive Substances 0.000 claims description 4
- 239000012928 buffer substance Substances 0.000 claims description 3
- 239000011148 porous material Substances 0.000 claims description 3
- 150000005846 sugar alcohols Chemical class 0.000 claims description 2
- 239000000470 constituent Substances 0.000 claims 2
- 230000002745 absorbent Effects 0.000 claims 1
- 239000002250 absorbent Substances 0.000 claims 1
- 230000003213 activating effect Effects 0.000 claims 1
- 239000007788 liquid Substances 0.000 abstract description 10
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 abstract description 5
- 108010000499 Thromboplastin Proteins 0.000 abstract description 5
- 102000002262 Thromboplastin Human genes 0.000 abstract description 5
- 230000004913 activation Effects 0.000 abstract description 4
- 230000006641 stabilisation Effects 0.000 abstract description 2
- 238000011105 stabilization Methods 0.000 abstract description 2
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 27
- 239000000872 buffer Substances 0.000 description 14
- 229960002885 histidine Drugs 0.000 description 14
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 13
- 239000001110 calcium chloride Substances 0.000 description 13
- 229910001628 calcium chloride Inorganic materials 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 238000000746 purification Methods 0.000 description 12
- 239000000203 mixture Substances 0.000 description 10
- 238000004587 chromatography analysis Methods 0.000 description 9
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 8
- 239000011734 sodium Substances 0.000 description 8
- 229910052938 sodium sulfate Inorganic materials 0.000 description 8
- 235000011152 sodium sulphate Nutrition 0.000 description 8
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 7
- 229930195725 Mannitol Natural products 0.000 description 7
- 238000009472 formulation Methods 0.000 description 7
- 239000000594 mannitol Substances 0.000 description 7
- 235000010355 mannitol Nutrition 0.000 description 7
- 239000012141 concentrate Substances 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 108010049003 Fibrinogen Proteins 0.000 description 5
- 102000008946 Fibrinogen Human genes 0.000 description 5
- 239000006227 byproduct Substances 0.000 description 5
- 229940012952 fibrinogen Drugs 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 238000003860 storage Methods 0.000 description 5
- 230000015271 coagulation Effects 0.000 description 4
- 238000005345 coagulation Methods 0.000 description 4
- 239000007858 starting material Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 4
- 108010080379 Fibrin Tissue Adhesive Proteins 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000001994 activation Methods 0.000 description 3
- 229940024606 amino acid Drugs 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 238000005341 cation exchange Methods 0.000 description 3
- 150000001768 cations Chemical class 0.000 description 3
- 239000012535 impurity Substances 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 3
- 239000010452 phosphate Substances 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 239000012541 Fractogel® Substances 0.000 description 2
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 229930064664 L-arginine Natural products 0.000 description 2
- 235000014852 L-arginine Nutrition 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 229940122388 Thrombin inhibitor Drugs 0.000 description 2
- 150000007513 acids Chemical class 0.000 description 2
- 238000005571 anion exchange chromatography Methods 0.000 description 2
- 235000011148 calcium chloride Nutrition 0.000 description 2
- 238000011210 chromatographic step Methods 0.000 description 2
- 238000011097 chromatography purification Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 238000011026 diafiltration Methods 0.000 description 2
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 2
- 239000006167 equilibration buffer Substances 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000000087 stabilizing effect Effects 0.000 description 2
- 239000003868 thrombin inhibitor Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 208000035404 Autolysis Diseases 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000004506 Blood Proteins Human genes 0.000 description 1
- 108010017384 Blood Proteins Proteins 0.000 description 1
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 1
- 206010057248 Cell death Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 108010071289 Factor XIII Proteins 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 239000008118 PEG 6000 Substances 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 241000125945 Protoparvovirus Species 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910001424 calcium ion Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000003467 diminishing effect Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229940012444 factor xiii Drugs 0.000 description 1
- 230000020764 fibrinolysis Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000012537 formulation buffer Substances 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000009928 pasteurization Methods 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920006316 polyvinylpyrrolidine Polymers 0.000 description 1
- 230000028043 self proteolysis Effects 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/14—Blood; Artificial blood
- A61K35/16—Blood plasma; Blood serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/482—Serine endopeptidases (3.4.21)
- A61K38/4833—Thrombin (3.4.21.5)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/02—Inorganic compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
- A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/043—Mixtures of macromolecular materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
- A61L24/10—Polypeptides; Proteins
- A61L24/106—Fibrin; Fibrinogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6429—Thrombin (3.4.21.5)
Definitions
- the invention relates to a thrombin preparation which is stable in the liquid state and is distinguished by high purity and virus safety, and to a process for its production.
- thrombin Since it became possible to produce thrombin commercially, several applications thereof have emerged. The main applications to be mentioned at present are, besides diagnostic purposes, the use as local hemostatic or as component of a tissue glue together with a fibrogen-containing component.
- the precondition for use of thrombin for medical purposes is that it can be made available to the clinician as a stable product which has high virus safety and contains minimal amounts of inactive byproducts or degradation products of thrombin or other factors.
- Japanese Patent Application No. 56-39782 discloses a process in which organic mono- or polycarboxylic acids and/or mono- or polyhydroxycarboxylic acids are employed to produce stable aqueous solutions of thrombin.
- Japanese Patent Application No. 57-18985 discloses albumin as thrombin stabilizer
- Japanese Patent Application No. 62-106028 discloses a buffer solution as stabilizer.
- European Patent Application 0 302 754 proposes a sugar and an amino acid, preferably in a concentration of from 1 to 10% by weight, as stabilizers for thrombin solutions.
- German Published Specification 31 22 926 further discloses a storable thrombin preparation which, besides sodium chloride, makes use of polyhydric alcohols with 3 to 6 carbon atoms, sulfur-free amino acids and polyethylene glycol for producing thrombin solutions.
- European Patent Application 0 221 700 describes a thrombin preparation which is buffered at a pH of from 5 to 8 and may contain, where appropriate, sodium chloride and a polyhydroxy compound.
- European Patent Application 0 439 156 discloses a process for producing a purified thrombin with a specific activity of more than 1600 U/mg, which can be employed for hemostasis. This entails using thromboplastin to activate prothrombin and employing an anion exchange chromatography and a cation exchange chromatography with support materials based on agarose.
- An “ultra-pure”, clear, colorless bovine thrombin with a specific activity of about 8000 to 11,000 NIH U/mg is described in U.S. Pat. No. 5,397,704.
- Thromboplastin from bovine lung is employed therein for prothrombin activation, and anion exchange chromatography and cation exchange chromatography are used for purification of the protein.
- thrombin preparation in which a prothrombin obtained from plasma or a plasma fraction is, after activation to thrombin without the addition of thromoplastin and, where appropriate, further processing steps, purified by a hydrophobic interaction chromatography and, where appropriate, subsequently the viruses are inactivated or removed.
- a further improvement in this process is possible if a cation exchange chromatography is additionally carried out before or after the hydrophobic interaction chromatography.
- the chromatographies in this case may be carried out as “positive” (binding of the thrombin) or as “negative” (binding of the impurities) chromatography.
- hydrophobic interaction chromatography HIC
- CEC cation exchange chromatography
- the production process of the invention is carried out in such a way that initially thrombin of low or moderate purity is produced. This can take place by adsorbing prothrombin from plasma or a plasma fraction on an ion exchanger.
- the prothrombin obtained in this way can then be subjected to a virus inactivation, e.g. by pasteurization or another known method, and, where appropriate, further processing steps, and then the thrombin can be activated by processes known per se without the addition of thromboplastin obtained from animal tissue.
- the subsequent hydrophobic interaction chromatography then reduces the concentration of concomitant plasma proteins, activated factors or their fragments, and of thrombin degradation products. This purification effect is further enhanced by the subsequent cation exchange chromatography.
- Elution of pure thrombin is followed by addition of suitable buffer substances to adjust the pH of the preparation to the range from 5 to 8, and stabilizers are added. It is also possible to add buffer substance(s) and stabilizers together.
- the adsorbent employed in the hydrophobic interaction chromatography which is known per se as chromatographic method, is a gel with coupled hydrophobic radicals.
- Particularly suitable hydrophobic radicals are in this case phenyl radicals or other ligands with a similar hydrophobicity.
- the cation exchanger preferably employed is a gel with high resolution for the various thrombin variants. Examples of suitable cation exchange gels are Fractogel EMD SO 3 (Merck, Darmstadt), Macro Prep 50S (Biorad, Kunststoff) or other cation exchangers complying with requirements in relation to purification and sterilizability.
- the thrombin solution obtained after chromatographic purification can then be subjected directly to virus inactivation or virus reduction such as, for example, filtration through small-pore membranes, which makes it possible effectively to remove even the smallest viruses while obtaining a high yield of thrombin.
- virus inactivation or reduction can, however, also take place before the chromatographic purification of thrombin if this facilitates the overall process (e.g. through removal of unwanted components or byproducts in the subsequent chromatography).
- a buffer should be used to adjust to a pH of about 5.0 to 8.0.
- a soluble calcium salt, sodium chloride, a sugar or a pure alcohol and/or an amino acid or else the salt of a mono- or polycarboxylic acid and/or the salt of a mono- or polyhydroxycarboxylic acid is added to the preparation. This results in good stabilities in the liquid and/or frozen state for a storage time of 12 months and more.
- Suitable substances for this purpose are compounds such as benzamidine or p-aminobenzamidine or other low to moderate affinity protease inhibitors. Addition of these low or moderate affinity inhibitors negligibly impairs the activity of thrombin in relation to substances such as fibrinogen, and thus also, for example, the later use as component of a tissue glue.
- thrombin preparations which can be stored in the liquid and/or frozen state for months or years and whose activity does not fall below 70-80% in this period.
- thrombin preparations which result in high stability at 4° C. for up to 24 months and more, as can be demonstrated in the coagulation test.
- Many of the thrombin preparations shown in Table 4 are also stable in the frozen state and, in most cases, show stability at room temperature for a period of 3 to 6 months.
- the stability at room temperature can be increased in particular by adding low or moderate affinity thrombin inhibitors such as, for example, benzamidine, p-aminobenzamidine or other protease or thrombin inhibitors, without this involving a significant decrease in the activity in relation to fibrinogen in the coagulation test.
- low or moderate affinity thrombin inhibitors such as, for example, benzamidine, p-aminobenzamidine or other protease or thrombin inhibitors
- the thrombin preparations produced by the described process can be employed inter alia as components of a fibrin glue which can be stored in the liquid or frozen state and consists of two components, e.g. of a thrombin-containing component and of a fibrinogen-containing component, or consisting of three components, e.g. of a thrombin-containing component, fibrinogen-containing component and factor XIII-containing component, as described inter alia in German Patent Application 198 53 033.1. It is moreover possible either for the thrombin preparation produced in this way to be mixed in situ with the other components, or, in the case of a three-component fibrin glue, for it to be mixed beforehand with one of the components before the third component is added. However, it is also possible to produce, lyophilized thrombin preparations using the process of the invention for therapeutic purposes, in which case a correspondingly high stability is observed in the liquid state after reconstitution.
- thrombin concentrates produced according to the invention can also be employed alone or in combination with carrier materials as agent for local stoppage of bleeding.
- the thrombin solution was mixed with 0.6 mol/l sodium sulfate and adsorbed onto a hydrophobic interaction chromatography (HIC) gel (in this case: Phenyl-Sepharose HP, manufacturer: Amersham Pharmacia, Freiburg, Germany) which had previously been equilibrated with buffer A (10 mmol/l Na phosphate pH 6.5) containing 0.6 mol/l sodium sulfate.
- buffer A 10 mmol/l Na phosphate pH 6.5
- the bound thrombin was eluted by a gradient with decreasing sodium sulfate content in buffer A. Impurities and thrombin fragments were to a large extent removed in the flow-through or in the wash fractions.
- the thrombin fraction was loaded without further treatment directly onto a cation exchange column (CEC; in this case: FRACTOGEL® EMD SO3, manufacturer: Merck, Darmstadt, Germany) equilibrated with buffer A, washed with equilibration buffer A and eluted by a gradient from 0 to 1.0 mol/l sodium chloride in buffer A.
- CEC cation exchange column
- equilibration buffer A eluted by a gradient from 0 to 1.0 mol/l sodium chloride in buffer A.
- final byproducts and thrombin fragments were removed so that the resulting ⁇ -thrombin eluate had a high specific purity of about 3500 lU/mg (protein determination by determining the absorption at 280 nm and using the conversion factor of 1.74 for a 0.1% strength solution in accordance with J. W. Fenton, II, M. J.
- thrombin solution was mixed with 0.6 mol/l sodium sulfate and adsorbed onto a hydrophobic interaction chromatography (HIC) gel (in this case: Phenyl-Sepharose HP, manufacturer: Amersham Pharmacia, Freiburg, Germany) which had previously been equilibrated with buffer B (10 mmol/l Na phosphate 0.1% PEG pH 6.5; (in this case PEG 6000, but other molecular weight ranges can also be employed)) containing 0.6 mol/l sodium sulfate.
- HIC hydrophobic interaction chromatography
- the bound thrombin was eluted by a gradient with decreasing sodium sulfate content in buffer B. Impurities and thrombin fragments were to a large extent removed in the flow-through or in the wash fractions.
- the thrombin fraction was loaded without further treatment directly onto a cation exchange column (CEC; in this case: FRACTOGEL® EMD SO3, manufacturer: Merck, Darmstadt, Germany) equilibrated with buffer C (10 mmol/Na phosphate, 166 mmol/l L-arginine pH 6.5), washed with equilibration buffer C and eluted by a gradient from 0 to 1.0 mol/l sodium chloride in buffer C. During the separation, final byproducts and thrombin fragments were removed so that the resulting ⁇ -thrombin eluate had a high specific purity of about 3300 lU/mg (cf. Table 2).
- CEC cation exchange column
- a thrombin purification was carried out as in Example 1 but with the difference that the buffer employed for the chromatography contains 20 mmol/l L-histidine in place of sodium phosphate.
- the results of purification with this modification are comparable to Example 1, but further processing to the final product may be simplified if in this case, for example, histidine is to be present as buffer substance.
- thrombin solutions were produced by diafiltration of the purified thrombin concentrates against the formulation buffer or by diafiltration against a basic buffer and adding the remaining additives, adjusting the pH and adjusting the thrombin concentration.
- Thrombin concentrations of about 1 to about 15,000 lU/ml can be produced in this way.
- thrombin formulations 1. 360 mmol/l NaCl, 40 mmol/l CaCl 2 , 5 mmol/l L-histidine ph 6.0 2. 360 mmol/l NaCl, 40 mmol/l CaCl 2 , 2% (w/v) mannitol, 5 mmol/l L-histidine pH 6.0 3.
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Abstract
Description
- This application is a division of application Ser. No. 09/809,021, filed Mar. 16, 2001, which claims priority to German Application No. DE 100 12 732.0, filed Mar. 18, 2000, both of which are incorporated herein by reference in their entirety.
- The invention relates to a thrombin preparation which is stable in the liquid state and is distinguished by high purity and virus safety, and to a process for its production.
- Since it became possible to produce thrombin commercially, several applications thereof have emerged. The main applications to be mentioned at present are, besides diagnostic purposes, the use as local hemostatic or as component of a tissue glue together with a fibrogen-containing component. The precondition for use of thrombin for medical purposes is that it can be made available to the clinician as a stable product which has high virus safety and contains minimal amounts of inactive byproducts or degradation products of thrombin or other factors.
- Numerous methods for stabilizing thrombin have already been proposed. Thus, Japanese Patent Application No. 56-39782 discloses a process in which organic mono- or polycarboxylic acids and/or mono- or polyhydroxycarboxylic acids are employed to produce stable aqueous solutions of thrombin. Japanese Patent Application No. 57-18985 discloses albumin as thrombin stabilizer, and Japanese Patent Application No. 62-106028 discloses a buffer solution as stabilizer. European Patent Application 0 302 754 proposes a sugar and an amino acid, preferably in a concentration of from 1 to 10% by weight, as stabilizers for thrombin solutions.
- German Published Specification 31 22 926 further discloses a storable thrombin preparation which, besides sodium chloride, makes use of polyhydric alcohols with 3 to 6 carbon atoms, sulfur-free amino acids and polyethylene glycol for producing thrombin solutions. Finally, European Patent Application 0 221 700 describes a thrombin preparation which is buffered at a pH of from 5 to 8 and may contain, where appropriate, sodium chloride and a polyhydroxy compound.
- Buffered and stabilized thrombin solutions are also disclosed in a publication by J. Chabbat et al. [J. Chabbat, M. Tellier, P. Porte and M. Steinbuch; Properties of a new fibrin glue stable in liquid state, Thromb. Res. 76: 525-533 (1994)].
- In addition, a publication by D. V. Brezniak, H. I. Hassouna and J. W. Fenton II [Blood Coagulation and Fibrinolysis, 6, 847-848 (1994)] has already described the effect of salts on the stability of dilute α-thrombin solutions. It was shown in this study that sodium chloride concentrations of 0.3 mol/l and above have a marked stabilizing effect in dilute thrombin solutions. It is stated that thrombin is stable in sodium chloride-containing solutions at 37° C. for about 2 weeks, and thus the stability is greater than in calcium chloride-containing solutions, which may possibly be explained by better thermal stability in relation to denaturation.
- Numerous patent applications have also described processes for producing high-purity thrombin preparations. Thus, European Patent Application 0 439 156 discloses a process for producing a purified thrombin with a specific activity of more than 1600 U/mg, which can be employed for hemostasis. This entails using thromboplastin to activate prothrombin and employing an anion exchange chromatography and a cation exchange chromatography with support materials based on agarose. An “ultra-pure”, clear, colorless bovine thrombin with a specific activity of about 8000 to 11,000 NIH U/mg is described in U.S. Pat. No. 5,397,704. Thromboplastin from bovine lung is employed therein for prothrombin activation, and anion exchange chromatography and cation exchange chromatography are used for purification of the protein.
- However, none of the processes disclosed to date allows a purified, calcium ion-containing, virus-safe thrombin preparation which is stable in the liquid state at 0° C. and higher temperatures, and whose thrombin activity after 12 months or more is still over 70-80% of the initial level, to be produced. The object therefore is to develop a process for producing such a thrombin preparation, the intention being to dispense with the use of thromboplastin for prothrombin activation for reasons of product safety. An additional object was to avoid high concentrations of polyols as added stabilizers, because this results in an unwanted increase in the viscosity of the preparation.
- It has now been found that this object is achieved by a process for producing a thrombin preparation in which a prothrombin obtained from plasma or a plasma fraction is, after activation to thrombin without the addition of thromoplastin and, where appropriate, further processing steps, purified by a hydrophobic interaction chromatography and, where appropriate, subsequently the viruses are inactivated or removed.
- A further improvement in this process is possible if a cation exchange chromatography is additionally carried out before or after the hydrophobic interaction chromatography. The chromatographies in this case may be carried out as “positive” (binding of the thrombin) or as “negative” (binding of the impurities) chromatography.
- Since an adequate purity of the thrombin solution employed is necessary to achieve high thrombin stability in the liquid state, a simple and improved process was sought for using high-purity thrombin with high virus safety. It is possible to use as basis for example the process described in European Patent Application 0 543 178 for producing a thrombin concentrate. However, other processes in which partially purified prothrombin is activated to thrombin in the presence of calcium salts can also be employed according to the invention as starting material for producing the thrombin preparation.
- If hydrophobic interaction chromatography (HIC) is employed alone or in combination with a cation exchange chromatography (CEC) for purifying thrombin, then effective and simple purification is achieved thereby. The sequence of these two chromatography processes is moreover as desired. If the chromatography is carried out initially with a hydrophobic support, the thrombin eluate can then be bound directly to the cation exchanger and eluted therefrom with a salt gradient. Combination of these two separation principles results in a thrombin preparation of high purity in a good yield of about 70% over the two purification steps. This simultaneously achieves good removal of byproducts such as activated or unactivated coagulation factors and of thrombin forms having little or no activity in the coagulation test (e.g. prothrombin, β-thrombin, γ-thrombin or other thrombin or prothrombin fragments). Combination of the two chromatography processes mentioned results in higher purity than use of ion exchange chromatography on its own.
- The production process of the invention is carried out in such a way that initially thrombin of low or moderate purity is produced. This can take place by adsorbing prothrombin from plasma or a plasma fraction on an ion exchanger. The prothrombin obtained in this way can then be subjected to a virus inactivation, e.g. by pasteurization or another known method, and, where appropriate, further processing steps, and then the thrombin can be activated by processes known per se without the addition of thromboplastin obtained from animal tissue. The subsequent hydrophobic interaction chromatography then reduces the concentration of concomitant plasma proteins, activated factors or their fragments, and of thrombin degradation products. This purification effect is further enhanced by the subsequent cation exchange chromatography.
- Elution of pure thrombin is followed by addition of suitable buffer substances to adjust the pH of the preparation to the range from 5 to 8, and stabilizers are added. It is also possible to add buffer substance(s) and stabilizers together.
- The adsorbent employed in the hydrophobic interaction chromatography, which is known per se as chromatographic method, is a gel with coupled hydrophobic radicals. Particularly suitable hydrophobic radicals are in this case phenyl radicals or other ligands with a similar hydrophobicity. The cation exchanger preferably employed is a gel with high resolution for the various thrombin variants. Examples of suitable cation exchange gels are Fractogel EMD SO3 (Merck, Darmstadt), Macro Prep 50S (Biorad, Munich) or other cation exchangers complying with requirements in relation to purification and sterilizability.
- The thrombin solution obtained after chromatographic purification can then be subjected directly to virus inactivation or virus reduction such as, for example, filtration through small-pore membranes, which makes it possible effectively to remove even the smallest viruses while obtaining a high yield of thrombin. Virus inactivation or reduction can, however, also take place before the chromatographic purification of thrombin if this facilitates the overall process (e.g. through removal of unwanted components or byproducts in the subsequent chromatography).
- For formulation of the thrombin preparation as a component, which is stable and storable in the liquid and, where appropriate, also in the frozen state, for use in a tissue glue or on its own as local hemostatic, a buffer should be used to adjust to a pH of about 5.0 to 8.0. To achieve the desired effect on use and for stabilization, then a soluble calcium salt, sodium chloride, a sugar or a pure alcohol and/or an amino acid or else the salt of a mono- or polycarboxylic acid and/or the salt of a mono- or polyhydroxycarboxylic acid is added to the preparation. This results in good stabilities in the liquid and/or frozen state for a storage time of 12 months and more.
- It has also emerged that addition of substances which inhibit noncovalently the thrombin activity in vitro can seemingly only increase the stability even further, especially at room temperature, by diminishing the autolysis of thrombin. Suitable substances for this purpose are compounds such as benzamidine or p-aminobenzamidine or other low to moderate affinity protease inhibitors. Addition of these low or moderate affinity inhibitors negligibly impairs the activity of thrombin in relation to substances such as fibrinogen, and thus also, for example, the later use as component of a tissue glue.
- It is possible via the process of the invention to produce thrombin preparations which can be stored in the liquid and/or frozen state for months or years and whose activity does not fall below 70-80% in this period.
- It is possible with the process of the invention to produce, even in the presence of calcium salts which reduce the thermal stability of thrombins [B. H. Landis, K. A. Koehler and J. W. Fenton II; Human Thrombins. J Biol chem 256: 4604-4610 (1981)], thrombin preparations which result in high stability at 4° C. for up to 24 months and more, as can be demonstrated in the coagulation test. Many of the thrombin preparations shown in Table 4 are also stable in the frozen state and, in most cases, show stability at room temperature for a period of 3 to 6 months. The stability at room temperature can be increased in particular by adding low or moderate affinity thrombin inhibitors such as, for example, benzamidine, p-aminobenzamidine or other protease or thrombin inhibitors, without this involving a significant decrease in the activity in relation to fibrinogen in the coagulation test.
- The thrombin preparations produced by the described process can be employed inter alia as components of a fibrin glue which can be stored in the liquid or frozen state and consists of two components, e.g. of a thrombin-containing component and of a fibrinogen-containing component, or consisting of three components, e.g. of a thrombin-containing component, fibrinogen-containing component and factor XIII-containing component, as described inter alia in German Patent Application 198 53 033.1. It is moreover possible either for the thrombin preparation produced in this way to be mixed in situ with the other components, or, in the case of a three-component fibrin glue, for it to be mixed beforehand with one of the components before the third component is added. However, it is also possible to produce, lyophilized thrombin preparations using the process of the invention for therapeutic purposes, in which case a correspondingly high stability is observed in the liquid state after reconstitution.
- Finally, the thrombin concentrates produced according to the invention can also be employed alone or in combination with carrier materials as agent for local stoppage of bleeding.
- The process of the invention is explained in more detail by the following examples:
- Starting from a thrombin concentrate of low or moderate purity, produced by known processes, two chromatography steps were carried out.
- Initially the thrombin solution was mixed with 0.6 mol/l sodium sulfate and adsorbed onto a hydrophobic interaction chromatography (HIC) gel (in this case: Phenyl-Sepharose HP, manufacturer: Amersham Pharmacia, Freiburg, Germany) which had previously been equilibrated with buffer A (10 mmol/l Na phosphate pH 6.5) containing 0.6 mol/l sodium sulfate. After washing with buffer A containing 0.6 mol/l sodium sulfate, the bound thrombin was eluted by a gradient with decreasing sodium sulfate content in buffer A. Impurities and thrombin fragments were to a large extent removed in the flow-through or in the wash fractions.
- The thrombin fraction was loaded without further treatment directly onto a cation exchange column (CEC; in this case: FRACTOGEL® EMD SO3, manufacturer: Merck, Darmstadt, Germany) equilibrated with buffer A, washed with equilibration buffer A and eluted by a gradient from 0 to 1.0 mol/l sodium chloride in buffer A. During the separation, final byproducts and thrombin fragments were removed so that the resulting α-thrombin eluate had a high specific purity of about 3500 lU/mg (protein determination by determining the absorption at 280 nm and using the conversion factor of 1.74 for a 0.1% strength solution in accordance with J. W. Fenton, II, M. J. Fasco, A. B. Stackrow, D. L. Aronson, A. M. Young and J. S. Finlayson, Human Thrombins. J Biol Chem 252; 3587-3598 (1977)). Table 1 shows the results of this thrombin purification and the resulting specific activity.
- At this stage, the thrombin can be stored in a chilled or deep-frozen state until processed further.
TABLE 1 Specific Absorption Protein* Activity activity Sample 280 nm (mg/ml) [IU/mi) (I U/mg) Thrombin, 13.78 7.92 6418 810 starting material HIC eluate 1.085 0.624 1372 2199 CEO eluate 6.65 3.822 13370 3498
*A280, 0.1% = 1.74
- Starting from a thrombin concentrate of moderate or low purity, two chromatography steps were carried out. Initially the thrombin solution was mixed with 0.6 mol/l sodium sulfate and adsorbed onto a hydrophobic interaction chromatography (HIC) gel (in this case: Phenyl-Sepharose HP, manufacturer: Amersham Pharmacia, Freiburg, Germany) which had previously been equilibrated with buffer B (10 mmol/l Na phosphate 0.1% PEG pH 6.5; (in this case PEG 6000, but other molecular weight ranges can also be employed)) containing 0.6 mol/l sodium sulfate. After washing with buffer B containing 0.6 mol/l sodium sulfate, the bound thrombin was eluted by a gradient with decreasing sodium sulfate content in buffer B. Impurities and thrombin fragments were to a large extent removed in the flow-through or in the wash fractions.
- The thrombin fraction was loaded without further treatment directly onto a cation exchange column (CEC; in this case: FRACTOGEL® EMD SO3, manufacturer: Merck, Darmstadt, Germany) equilibrated with buffer C (10 mmol/Na phosphate, 166 mmol/l L-arginine pH 6.5), washed with equilibration buffer C and eluted by a gradient from 0 to 1.0 mol/l sodium chloride in buffer C. During the separation, final byproducts and thrombin fragments were removed so that the resulting α-thrombin eluate had a high specific purity of about 3300 lU/mg (cf. Table 2).
- At this stage, the thrombin obtained can be stored in a chilled or deep-frozen state until processed further.
TABLE 2 Specific Absorption Protein* Activity activity Sample 280 nm (mg/ml) [IU/ml) (IU/mg) Thrombin, 12.49 7.178 5895 821 starting material HIC eluate 2.042 1.174 2696 2296 CEC eluate 8.03 4.615 15,150 3283
*A280, 0.1% = 1.74
- A thrombin purification was carried out as in Example 1 but with the difference that the buffer employed for the chromatography contains 20 mmol/l L-histidine in place of sodium phosphate. The results of purification with this modification are comparable to Example 1, but further processing to the final product may be simplified if in this case, for example, histidine is to be present as buffer substance.
- Starting from a thrombin eluate purified as in Examples 1 to 3 and after hydrophobic interaction chromatography and cation exchange chromatography, a filtration was carried out on a membrane with a small pore size (e.g. PLANOVA™ 15 nm). Even small viruses such as parvoviruses can be effectively removed with this membrane. It was found that on use of the purified thrombin as starting material, very good yields in terms of thrombin activity and protein were obtained, with a good filtration rate (see Table 3). This process is therefore suitable for producing a thrombin concentrate with high virus reduction factors.
TABLE 3 Filtration of 123 ml of purified thrombin through a Planova ™ 15 nm module (0.001 m2) Sample Thrombin activity, total Protein, total* Before filtration 800,240 IU 245.3 mg After filtration 797,960 IU 239.0 mg Yield 99.7% 97.4%
*A280, 0.1% = 1.74
- Starting from thrombin purified by chromatography, various formulations were produced and stored at temperatures of −20° C., 4° C., 20-25° C. and, in some cases, also at 37° C. These thrombin solutions were produced by diafiltration of the purified thrombin concentrates against the formulation buffer or by diafiltration against a basic buffer and adding the remaining additives, adjusting the pH and adjusting the thrombin concentration. Thrombin concentrations of about 1 to about 15,000 lU/ml can be produced in this way.
- The stability of the formulations was tested by determining the thrombin activity in a coagulation test with fibrinogen as substrate. Table 4 shows a selection of the formulations of the invention and their stabilizer composition, and Table 5 shows the corresponding stability data at up to three temperatures.
TABLE 4 Composition of thrombin formulations 1. 360 mmol/l NaCl, 40 mmol/l CaCl2, 5 mmol/l L-histidine ph 6.0 2. 360 mmol/l NaCl, 40 mmol/l CaCl2, 2% (w/v) mannitol, 5 mmol/l L-histidine pH 6.0 3. 150 mmol/l NaCl, 40 mmol/l CaCl2, 2% (w/v) mannitol, 5 mmol/l L-histidine pH 6.0 4. 90 mmol/l NaCl, 40 mmol/l CaCl2, 100 mmol/l Na succinate, 5 mmol/l L-histidine pH 6.0 5. 90 mmol/l NaCl, 40 mmol/l CaCl2, 100 mmol/l Na succinate, 2% (w/v) mannitol, 5 mmol/l L-histidine pH 6.0 6. 150 mmol/l NaCl, 40 mmol/l CaCl2, 100 mmol/l Na succinate, 5 mmol/l L-histidine pH 6.0 7. 90 mmol/l NaCl, 40 mmol/l CaCl2, 50 mmol/l Na lactate, 2% (w/v) mannitol, 5 mmol/l L-histidine pH 6.0 8. 90 mmol/l NaCl, 40 mmol/l CaCl2, 2% (w/v) mannitol, 10 mmol/l p-aminobenzamidine, 5 mmol/l L-histidine pH 6.0 9. 90 mmol/l NaCl, 40 mmol/l CaCl2, 2% (w/v) mannitol, 10 mmol benzamidine, 5 mmol/l L-histidine pH 6.0 10. 90 mmol/l NaCl, 40 mmol/l CaCl2, 4% (w/v) HSA, 5 mmol/l L-histidine pH 6.0 11. 90 mmol/l NaCl, 40 mmol/l CaCl2, 1% (w/v) mannitol, 142 mmol/l L-arginine, 5 mmol/l L-histidine pH 6.0 12. 90 mmol/l NaCl, 40 mmol CaCl2, 100 mmol/l Na succinate, 0.1% polyvinylpyrrolidone (K15), 5 mmol/l L-histidine pH 6.0. -
TABLE 5 Stability of thrombin in various formulations at 4° C., −20° C., 20-25° C., Storage time Mixture (month) 1 2 3 4 5 6 7 8 9 10 11 12 Thrombin activity (% of baseline value), storage temperature: 4° C. 0 100 100 100 100 100 100 100 100 100 100 100 100 1 101.5 95.8 98.9 94.0 100.4 100.8 110.6 102 98.0 106.8 108.3 98.0 3 98.9 109.8 103.4 103.1 108.8 104.5 101.6 95.6 91.5 98.5 102.3 103.9 6 100.5 117.5 88.4 97.3 99.0 98.2 108.1 97.6 95.2 103.0 99.4 101.9 9 97.5 112.9 95.6 94.2 91.9 91.6 122.7 102.2 99.6 102.2 91.5 108.0 12 100.2 116.5 93.6 92.4 105.8 106.4 117.2 100.2 97.8 101.2 93.5 103.8 18 89.7 101.7 — 94.7 112.7 — — 89.2 95.8 104.1 24 100.5 86.8 95.5 100 94.3 89.2 96.2 Thrombin activity (% of baseline value), storage temperature: −20° C. 0 100 100 100 100 100 100 100 100 1 103.6 100.4 88.6 94.3 94.9 47.1 111.0 106.5 3 97.5 92.6 104.2 93.0 93.9 91.5 103.0 101.3 6 99.5 92.7 101.3 99.7 98.2 68.6 107.3 96.4 9 100.3 81.3 92.9 89.2 87.6 92.1 108.0 95.3 12 94.3 100.5 95.3 104.4 98.5 79.2 104.0 95.1 18 94.3 — 92.7 — 99.3 99.8 24 99.0 90.7 100.7 97.2 104.2 100.2 Thrombin activity (% of baseline value), storage temperature: 20-25° C. 0 100 100 100 100 100 100 100 100 100 100 100 100 1 105.6 92.4 90.9 91.8 92.6 94.3 95.5 101.7 99.8 107.3 99.4 81.1 3 92.3 88.2 86.9 88.4 80.0 77.9 75.8 95.4 96.3 84.2 98.9 77.0 6 85.6 66.3 66.8 80.4 71.4 68.6 75.3 92.8 89.0 69.4 84.8 60.6 9 72.1 58.3 58.8 75.9 51.8 50.7 59.1 96.1 91.9 48.8 74.4 53.3 12 64.2 50.1 51.5 65.3 43.7 43.9 42.8 100.9 90.6 42.9 64.7 46.6 18 — — 24 90.1 82.4
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US09/809,021 Expired - Fee Related US7351561B2 (en) | 2000-03-18 | 2001-03-16 | Thrombin preparations and process for their production |
US11/385,713 Expired - Fee Related US8012728B2 (en) | 2000-03-18 | 2006-03-22 | Thrombin preparations and process for their production |
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US09/809,021 Expired - Fee Related US7351561B2 (en) | 2000-03-18 | 2001-03-16 | Thrombin preparations and process for their production |
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US (2) | US7351561B2 (en) |
EP (1) | EP1136084B1 (en) |
JP (1) | JP5577005B2 (en) |
KR (1) | KR100916131B1 (en) |
AU (2) | AU784992B2 (en) |
CA (2) | CA2340863C (en) |
DE (1) | DE10012732A1 (en) |
ES (1) | ES2654312T3 (en) |
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- 2001-02-20 ES ES01103992.2T patent/ES2654312T3/en not_active Expired - Lifetime
- 2001-03-15 CA CA2340863A patent/CA2340863C/en not_active Expired - Fee Related
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- 2001-03-16 JP JP2001076700A patent/JP5577005B2/en not_active Expired - Fee Related
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US20100047834A1 (en) * | 2006-12-21 | 2010-02-25 | Sekisui Medical Co., Ltd. | Method for stabilizing alpha-thrombin in thrombin-containing solution |
US8580532B2 (en) | 2006-12-21 | 2013-11-12 | Sekisui Medical Co., Ltd. | Method for stabilizing α-thrombin in thrombin-containing solution |
US20100086953A1 (en) * | 2008-10-02 | 2010-04-08 | Siemens Healthcare Diagnostics Products Gmbh | Blood Coagulation Assays |
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Also Published As
Publication number | Publication date |
---|---|
DE10012732A1 (en) | 2001-09-20 |
US20010033837A1 (en) | 2001-10-25 |
ES2654312T3 (en) | 2018-02-13 |
AU784992B2 (en) | 2006-08-17 |
US7351561B2 (en) | 2008-04-01 |
CA2340863C (en) | 2012-02-07 |
CA2735316A1 (en) | 2001-09-18 |
AU2006203509B2 (en) | 2010-03-04 |
CA2735316C (en) | 2015-04-28 |
AU2006203509B9 (en) | 2010-04-01 |
AU2804201A (en) | 2001-09-20 |
EP1136084A1 (en) | 2001-09-26 |
CA2340863A1 (en) | 2001-09-18 |
EP1136084B1 (en) | 2017-10-04 |
AU2006203509A1 (en) | 2006-09-07 |
US8012728B2 (en) | 2011-09-06 |
JP5577005B2 (en) | 2014-08-20 |
KR100916131B1 (en) | 2009-09-08 |
KR20010090000A (en) | 2001-10-17 |
JP2001261574A (en) | 2001-09-26 |
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